Tremendous efforts have been made to derive embryonic stem cells (ESC) from various species, primarily in mammals. Developing these cell types with self-renewal and differentiation capacities in avian species presents a double interest: 1) fundamental, to study early development and to decipher the mechanisms involved in the maintenance of pluripotency in vitro. 2) biotechnological, to analyze viral replication and to produce specific vaccines instead of using embryonated eggs. So far, only chicken ESC lines and one proprietary duck ESC line have been established and all of them were derived in the presence of serum. The small number of available stem cell lines limits the possibility of comparing avian species among themselves and with mammalian species. Their derivation in media containing serum decreases their potential use for pharmaceutical or veterinary purposes. We have therefore chosen to derive, in chemically defined media, new cell lines from stage X Pekin duck embryos. We have established different independent isolates which can be established in long term culture. The cells maintain the transcriptional signature such as OCT4, NANOG, TERT and ALPL gene transcription and physiological markers of ESC (telomerase activity, Alkaline phosphatase activity) and plasticity of differentiation. These populations then share most of the characteristics expected of pluripotent stem cells (PSCs). Therefore, these new duck PSCs can provide a working basis to analyze the mechanisms of maintenance of pluripotency in avian species by comparing them with already existing chicken cells. Moreover, it has been shown that certain endogenous retroviral elements, usually silent in most cell types, can be transcriptionally reactivated in pluripotent cells. This property remains a major drawback for their use as a substrate for the production of human and veterinary vaccines. We therefore analyzed the activity of reverse transcriptase in the supernatant of different chicken and duck cell lines. We have shown that the new duck ES lines exhibit a very low RT activity, 3 log below the activities measured in the chicken ES cell lines. We then tested the susceptibility of these duck ES cells to influenza viruses. We have shown that duck ES cells, but not duck embryonic fibroblasts, can replicate both human or avian influenza viruses as much as chicken ES cells. These Duck stem cells can therefore be a perfect substrate for producing vaccines under greater safety conditions than those with chicken embryos.